In Gram negative bacteria, production of bis-(3’,5’)-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides (EPS) and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium.The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.
Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors / D. Antoniani, P. Bocci, A. Maciag, N. Raffaelli, P. Landini. - In: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. - ISSN 0175-7598. - 85:4(2010), pp. 1095-1104.
|Titolo:||Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors|
ANTONIANI, DAVIDE (Primo)
LANDINI, PAOLO (Ultimo)
|Parole Chiave:||Antimicrobial drugs; Biofilm formation; C-di-GMP; Diguanylate cyclase; High-throughput screening; Sulfathiazole|
|Settore Scientifico Disciplinare:||Settore BIO/19 - Microbiologia Generale|
Settore BIO/10 - Biochimica
Settore BIO/11 - Biologia Molecolare
|Data di pubblicazione:||2010|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1007/s00253-009-2199-x|
|Appare nelle tipologie:||01 - Articolo su periodico|